Recombinant DNA technology is now widely used for both research and commercial protein production. An essential tool of this technology is the plasmid, a double-stranded DNA molecule that can replicate (autonomously or by chromosomal integration) in one or more species of host cell. A DNA sequence of interest can be inserted into a plasmid and replicated in large quantities. If a sequence encoding a protein is operably linked to a transcription promoter, the sequence can be expressed and the encoded protein can be recovered from the cell.
DNA sequences of interest are commonly joined to plasmids by cutting both pieces of DNA with a restriction endonuclease to provide complementary ends that are then enzymatically ligated together. If the sequences of the two pieces do not permit the use of a single restriction endonuclease, small oligonucleotide adapters can be used to join the free ends.
Advances in recombinant DNA technology, including the use of automation, have resulted in the rapid identification and production of novel DNA sequences. To further characterize these sequences it is necessary to express them and study the properties of the encoded proteins. Expression generally requires the precise joining of DNA sequences within expression vectors to maintain the functional relationships between genetic elements (e.g., open reading frame, promoter function, etc.). Current methods, which rely primarily on the use of restriction enzymes, can be problematic when it is necessary to rapidly express a large number of sequences and analyze their products because these methods can require extensive and time-consuming manipulation of sequences to obtain the desired junctions. There is thus a need in the art for improved methods of plasmid construction. Toward this end, the present invention provides a standardized plasmid into which a variety of DNA sequences can be readily inserted and subsequently expressed, as well as related cloning methods and other improvements.